Sterilization and filtration of peptide compositions

ABSTRACT

Methods and devices for sterilizing viscous peptide compositions which have shear thinning rheological properties at high concentrations.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) of U.S.provisional patent application Ser. No. 61/950,536, filed Mar. 10, 2014,which application is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

This application makes reference to a sequence listing submitted inelectronic form as an ASCII .txt file named “2004837-0044Sequences.txt”. The .txt file was generated on Mar. 9, 2015 and is 1 kbin size.

BACKGROUND

Peptide agents with the ability to self-assemble into gel structureshave a wide variety of uses in therapeutic and research contexts. Onesuch peptide agent, for example, a synthetic, 16-amino acid polypeptidewith a repeating sequence of arginine, alanine, and aspartic acid (i.e.,RADARADARADARADA [SEQ ID NO:1], also known as “RADA16”), is commerciallyavailable under the trade names PuraStat, PuraMatrix®, and PuraMatrixGMP® from 3-D Matrix Medical Technology, and has demonstrated utility ina wide range of laboratory and clinical applications, including cellculture, drug delivery, accelerated cartilage and bone growth, andregeneration of CNS, soft tissue, and cardiac muscle, and furthermore asa matrix, scaffold, or tether that can be associated with one or moredetectable agents, biologically active agents, cells, and/or cellularcomponents.

SUMMARY

The present invention provides, among other things, methods for handlingpeptide compositions and technologies relating thereto. Teachingsprovided herein may be particularly applicable to high-viscosity peptidecompositions, and/or compositions of self-assembling peptides.

Among other things, the present disclosure demonstrates that certainpeptide compositions (e.g., compositions of particular peptides, atparticular concentrations, and/or having particular rheologicalproperties) have certain characteristics and/or may not be amenable tocertain handling and/or processing steps such as, for example,filtration (e.g., sterilizing filtration).

The present disclosure also demonstrates that certain particular peptidecompositions are surprisingly stable to one or more treatments (e.g.,heat treatment, as is applied in autoclave procedures) that damage manypeptide compositions.

Thus, the present disclosure provides a variety of technologies relevantto processing of peptide compositions, and particularly tosterilization.

In some embodiments, the present disclosure demonstrates that particularpeptide compositions may have one or more useful and/or surprisingcharacteristics (e.g., resistance to damage from heat treatment,rheological responsiveness to and/or recovery from application of shearstress, etc).

The present disclosure provides, among other things, systems forsterilizing peptide compositions, and/or systems for determiningappropriate such systems for application to particular peptidecompositions.

In some embodiments, particular peptide compositions may be defined, forexample, by one or more features selected from the group consisting of:peptide sequence, peptide concentration, viscosity, stiffness,sensitivity to heat treatment, rheological responsiveness to applicationof shear stress, rheological recovery from application of shear stress,etc).

Among other things, the present disclosure provides certain peptidecompositions that may be sterilized by autoclave treatment.

In some embodiments, the present disclosure provides certaintechnologies for achieving filtration of certain peptide compositions,and particularly for altering rheological properties of peptidecompositions (as defined by identity and sequence of the peptide) sothat they are rendered amenable to filtration. For example, in someembodiments, viscosity of peptide compositions to be filtered may bereduced prior to filtration. In some embodiments, shear stress may beapplied to peptide compositions, so that rheological properties may bealtered. For example, viscosity and/or stiffness of a peptidecomposition may be reduced prior to filtration; in some embodiments,such a reduction is temporary.

In some embodiments, provided technologies enable filtration of peptidecompositions at higher concentrations than is feasible with conventionalfiltration techniques. For example, technologies described herein permitRADA16 to be filtered, and particularly to be sterilized by filtration,at concentrations higher than 2.5% in accordance.

In some particular embodiments, the present disclosure provides a methodfor sterilizing a liquid peptide composition whose sequence comprises aseries of repeating units of IEIK comprising subjecting the compositionto autoclave treatment. In some embodiments, a method does not involvesterilizing filtration.

In some embodiments, the present disclosure provides a method forsterilizing a liquid peptide composition whose sequence comprises aseries of repeating units of IEIK comprising subjecting the compositionto heat treatment. In some embodiments, the heat treatment performs atabout 121° C. for about 25 min.

In some embodiments, the present disclosure provides a method forsterilizing a liquid peptide composition having an initial storagemodulus within the range of about 300 to about 5,000 Pa at 1 Pa ofoscillation stress, the method comprising steps of subjecting thecomposition to high shear stress so that storage modulus of thecomposition is temporarily reduced to a level within a range of about0.01% to 80% of the initial storage modulus, and subjecting thecomposition to filtration while its viscosity is at the reduced level.

In some embodiments, the step of subjecting the composition to highshear stress utilizes at least one shear-thinning unit.

In some embodiments, at least one shear-thinning unit is or comprises atleast one needle. In some embodiments, at least one needle is at least10 mm long. In some embodiments, at least one needle has a gauge withinthe range of about 25 to about 35.

In some embodiments, at least one shear-thinning unit is or comprises atleast one screen with micro- or nano-sized holes. In some embodiments,micro- or nano-sized holes have a largest dimension within a range ofabout 0.5 μm to about 200 μm. In some embodiments, a pinch between holesis about 5 μm to about 10 mm. In some embodiments, a screen is made atleast in part of a material selected from the group consisting ofstainless-steel, tungsten, titanium, silicon, ceramic, plastic, andcombination thereof. In some embodiments, thickness of the screen isabout 10 μm to about 10 mm.

In some embodiments, at least one shear-thinning unit is or comprises atleast one membrane with micro- or nano-sized pores. In some embodiments,the pores gave a size with a range of about 0.45 μm to about 120 μm.

In some embodiments, high shear stress for sterilization is with a rangeof about 30 to about 200 Pa.

In some embodiments, a liquid peptide composition comprises RADA16,IEIK13, or KLD12.

In some embodiments, a liquid peptide composition is pressurized priorto filtration. In some embodiments, a peptide liquid composition isfurther stored the under vacuum.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A and 1B show exemplary mass spectrometry analysis of RADA16before and after autoclave treatment, to assess heat sensitivity. FIG.1A depicts mass spectrometry before autoclave treatment. FIG. 1B depictsmass spectrometry after autoclave treatment. FIG. 1C illustratesexemplary RADA16 molecular structure; in the particular peptidecomposition that was analyzed, the protein was composed ofRADARADARADARADA where the N-terminus and C-terminus are protected byacetyl and amino groups.

FIGS. 2A and 2B show exemplary mass spectrometry analysis of IEIK13before and after autoclave treatment, to assess. FIG. 2A depicts massspectrometry before autoclave treatment. FIG. 2B depicts massspectrometry after autoclave treatment. FIG. 2C illustrates exemplaryIEIK13 molecular structure; in the particular peptide composition thatwas analyzed, the protein was composed of IEIKIEIKIEIKI, where theN-terminus and C-terminus are protected by acetyl and amino groups.

FIGS. 3A and 3B show exemplary mass spectrometry of KLD12 before andafter autoclave treatment, to assess heat sensitivity. FIG. 3A depictsmass spectrometry before autoclave treatment. FIG. 3B depicts massspectrometry after autoclave treatment. FIG. 3C illustrates exemplaryKLD12 molecular structure; in the particular peptide composition thatwas analyzed, the protein was composed of KLDLKLDKKLDL, where theN-terminus and C-terminus are protected by acetyl and amino groups.

FIG. 4 shows exemplary time sweep tests of RADA16 and IEIK13 before andafter autoclave treatment.

FIG. 5 provides a picture of peptides and devices needed for filteringviscous peptide solutions, for example, RADA16, KLD12, and IEIK13. Shearstress was applied through 30-gauge needle to peptide solutions(middle), so that the peptide solutions were filtered (right).

FIG. 6 shows exemplary time sweep tests of 1% and 2.5% RADA16 performedat 1 Pa and 10 rad/s. RADA16 was injected through 30-gauge needle sothat applied shear stress reduced stiffness of the peptides. Themeasurements were started 1 minute after the injection. FIG. 7 showsexemplary time sweep tests 1% and 2.5% KLD12 performed at 1 Pa and 10rad/s. KLD12 was injected through 30-gauge needle so that the appliedshear stress reduced stiffness of the peptides. The measurements werestarted 1 minute after the injection.

FIG. 8 shows exemplary time sweep tests 2.5% IEIK13 performed at 1 Paand 10 rad/s. IEIK13 was injected through 30-gauge needle so that theapplied shear stress reduced stiffness of the peptides. The measurementswere started 1 minute after the injection.

FIG. 9 shows an exemplary flow viscosity test performed from 0.003 to1000 l/sec of shear rate on 2.5% RADA16 solution.

FIG. 10 shows an exemplary flow viscosity test performed from 0.003 to1000 l/sec of shear rate on 1.5% IEIK13 solution.

FIG. 11 shows exemplary viscosity measurements of 2.5% RADA16 as afunction of time to demonstrate viscosity recovery. At time=0, shearstress was applied to the peptides, so that the viscosity was reduced.The horizontal line indicates the original viscosity of 2.5% RADA16.

FIG. 12 shows exemplary viscosity measurements of 1.5% IEIK13 as afunction of time to demonstrate viscosity recovery. At time=0, shearstress was applied to the peptides, so that the viscosity was reduced.The horizontal line indicates the original viscosity of 1.5% IEIK13.

FIG. 13 shows exemplary devices for filtering viscous peptide solutions,for example, RADA16, KLD12, and IEIK13. Shear stress was applied througha shear-thinning unit with multiple pores. Peptide solution wasdispensed with a syringe on the top (i). Peptide solution passed throughthe first shear-thinning chamber (25 mm filter holder, Millipore (ii))where a shear-thinning unit with pores or holes was inserted to reducethe viscosity of peptide solution temporarily. Peptide solution thenpassed into second filtering chamber (25 mm filter holder, Millipore(iii)) where a filtering membrane was inserted to sterilize peptidesolutions or remove particulates from peptide solutions. Filteredsolution was received in a bottle (iv) for output. A high pressuredispenser was connected to the dispensing syringe (v). High pressurenitrogen gas was connected to the high pressure dispenser (vii).

FIG. 14 shows visual observation of viscosity after applying shearstress to 2.5% KLD12 solution and 1.5% IEIK13 solution. The top rowincludes pictures of 2.5% KLD. The bottom row includes pictures of 1.5%IEIK13. The solutions in the most left column stay on the top of thevials. The solutions in the most right column (after applying shearstress) have low viscosity, so that most materials are located at thebottom of vials.

FIGS. 15A, 15B, 15C, and 15D show materials and devices (a micro- ornano-hole screen) for filtering viscous peptide solutions such asRADA16, KLD12, and IEIK13. FIGS. 15A, 15B and 15C show features of anexemplary shear thinning unit, a micro-hole screen, which may be used inthe device shown in FIG. 13. Holes were generated by laser-drillingtechnology. Such a screen may be inserted in the first chamber to reduceviscosity of peptide solutions before actual filtration through themembrane in the second chamber. FIG. 15D shows visual observation ofviscosity after applying shear stress to 2.5% KLD12 using a micro- ornano-hole screen

DEFINITIONS

The term “agent” as used herein may refer to a compound or entity of anychemical class including, for example, polypeptides, nucleic acids,saccharides, lipids, small molecules, metals, or combinations thereof.In some embodiments, an agent is or comprises a natural product in thatit is found in and/or is obtained from nature. In some embodiments, anagent is or comprises one or more entities that is man-made in that itis designed, engineered, and/or produced through action of the hand ofman and/or is not found in nature. In some embodiments, an agent may beutilized in isolated or pure form; in some embodiments, an agent may beutilized in crude form. In some embodiments, potential agents areprovided as collections or libraries, for example that may be screenedto identify or characterize active agents within them. Some particularembodiments of agents that may be utilized in accordance with thepresent invention include small molecules, antibodies, antibodyfragments, aptamers, nucleic acids (e.g., siRNAs, shRNAs, DNA/RNAhybrids, antisense oligonucleotides, ribozymes), peptides, peptidemimetics, etc. In some embodiments, an agent is or comprises a polymer.In some embodiments, an agent is not a polymer and/or is substantiallyfree of any polymer. In some embodiments, an agent contains at least onepolymeric moiety. In some embodiments, an agent lacks or issubstantially free of any polymeric moiety.

As used herein, the term “amino acid,” in its broadest sense, refers toany compound and/or substance that can be incorporated into apolypeptide chain, e.g., through formation of one or more peptide bonds.In some embodiments, an amino acid has the general structureH2N—C(H)(R)—COOH. In some embodiments, an amino acid is anaturally-occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a D-aminoacid; in some embodiments, an amino acid is an L-amino acid. “Standardamino acid” refers to any of the twenty standard L-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.In some embodiments, an amino acid, including a carboxy- and/oramino-terminal amino acid in a polypeptide, can contain a structuralmodification as compared with the general structure above. For example,in some embodiments, an amino acid may be modified by methylation,amidation, acetylation, and/or substitution as compared with the generalstructure. In some embodiments, such modification may, for example,alter the circulating half-life of a polypeptide containing the modifiedamino acid as compared with one containing an otherwise identicalunmodified amino acid. In some embodiments, such modification does notsignificantly alter a relevant activity of a polypeptide containing themodified amino acid, as compared with one containing an otherwiseidentical unmodified amino acid. As will be clear from context, in someembodiments, the term “amino acid” is used to refer to a free aminoacid; in some embodiments it is used to refer to an amino acid residueof a polypeptide.

As used herein, the term “approximately” or “about,” as applied to oneor more values of interest, refers to a value that is similar to astated reference value. In certain embodiments, the term “approximately”or “about” refers to a range of values that fall within 25%, 20%, 19%,18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, or less in either direction (greater than or less than) of thestated reference value unless otherwise stated or otherwise evident fromthe context (except where such number would exceed 100% of a possiblevalue).

Two events or entities are “associated” with one another, as that termis used herein, if the presence, level and/or form of one is correlatedwith that of the other. For example, a particular entity (e.g.,polypeptide, genetic signature, metabolite, etc) is considered to beassociated with a particular disease, disorder, or condition, if itspresence, level and/or form correlates with incidence of and/orsusceptibility to the disease, disorder, or condition (e.g., across arelevant population). In some embodiments, two or more entities arephysically “associated” with one another if they interact, directly orindirectly, so that they are and/or remain in physical proximity withone another. In some embodiments, two or more entities that arephysically associated with one another are covalently linked to oneanother; in some embodiments, two or more entities that are physicallyassociated with one another are not covalently linked to one another butare non-covalently associated, for example by means of hydrogen bonds,van der Waals interaction, hydrophobic interactions, magnetism, andcombinations thereof.

The term “comparable” is used herein to describe two (or more) sets ofconditions, circumstances, individuals, or populations that aresufficiently similar to one another to permit comparison of resultsobtained or phenomena observed. In some embodiments, comparable sets ofconditions, circumstances, individuals, or populations are characterizedby a plurality of substantially identical features and one or a smallnumber of varied features. Those of ordinary skill in the art willappreciate that sets of circumstances, individuals, or populations arecomparable to one another when characterized by a sufficient number andtype of substantially identical features to warrant a reasonableconclusion that differences in results obtained or phenomena observedunder or with different sets of circumstances, individuals, orpopulations are caused by or indicative of the variation in thosefeatures that are varied. Those skilled in the art will appreciate thatrelative language used herein (e.g., enhanced, activated, reduced,inhibited, etc) will typically refer to comparisons made undercomparable conditions.)

Certain methodologies described herein include a step of “determining”.Those of ordinary skill in the art, reading the present specification,will appreciate that such “determining” can utilize or be accomplishedthrough use of any of a variety of techniques available to those skilledin the art, including for example specific techniques explicitlyreferred to herein. In some embodiments, determining involvesmanipulation of a physical sample. In some embodiments, determininginvolves consideration and/or manipulation of data or information, forexample utilizing a computer or other processing unit adapted to performa relevant analysis. In some embodiments, determining involves receivingrelevant information and/or materials from a source. In someembodiments, determining involves comparing one or more features of asample or entity to a comparable reference.

The term “gel” as used herein refers to viscoelastic materials whoserheological properties distinguish them from solutions, solids, etc. Insome embodiments, a composition is considered to be a gel if its storagemodulus (G′) is larger than its modulus (G″). In some embodiments, acomposition is considered to be a gel if there are chemical or physicalcross-linked networks in solution, which is distinguished from entangledmolecules in viscous solution.

The term “in vitro” as used herein refers to events that occur in anartificial environment, e.g., in a test tube or reaction vessel, in cellculture, etc., rather than within a multi-cellular organism.

The term “in vivo” as used herein refers to events that occur within amulti-cellular organism, such as a human and a non-human animal. In thecontext of cell-based systems, the term may be used to refer to eventsthat occur within a living cell (as opposed to, for example, in vitrosystems).

The term “peptide” as used herein refers to a polypeptide that istypically relatively short, for example having a length of less thanabout 100 amino acids, less than about 50 amino acids, less than 20amino acids, or less than 10 amino acids.

The term “polypeptide” as used herein refers to any polymeric chain ofamino acids. In some embodiments, a polypeptide has an amino acidsequence that occurs in nature. In some embodiments, a polypeptide hasan amino acid sequence that does not occur in nature. In someembodiments, a polypeptide has an amino acid sequence that is engineeredin that it is designed and/or produced through action of the hand ofman. In some embodiments, a polypeptide may comprise or consist ofnatural amino acids, non-natural amino acids, or both. In someembodiments, a polypeptide may comprise or consist of only natural aminoacids or only non-natural amino acids. In some embodiments, apolypeptide may comprise D-amino acids, L-amino acids, or both. In someembodiments, a polypeptide may comprise only D-amino acids. In someembodiments, a polypeptide may comprise only L-amino acids. In someembodiments, a polypeptide may include one or more pendant groups orother modifications, e.g., modifying or attached to one or more aminoacid side chains, at the polypeptide's N-terminus, at the polypeptide'sC-terminus, or any combination thereof. In some embodiments, suchpendant groups or modifications may be selected from the groupconsisting of acetylation, amidation, lipidation, methylation,pegylation, etc., including combinations thereof. In some embodiments, apolypeptide may be cyclic, and/or may comprise a cyclic portion. In someembodiments, a polypeptide is not cyclic and/or does not comprise anycyclic portion. In some embodiments, a polypeptide is linear. In someembodiments, a polypeptide may be or comprise a stapled polypeptide. Insome embodiments, the term “polypeptide” may be appended to a name of areference polypeptide, activity, or structure; in such instances it isused herein to refer to polypeptides that share the relevant activity orstructure and thus can be considered to be members of the same class orfamily of polypeptides. For each such class, the present specificationprovides and/or those skilled in the art will be aware of exemplarypolypeptides within the class whose amino acid sequences and/orfunctions are known; in some embodiments, such exemplary polypeptidesare reference polypeptides for the polypeptide class or family. In someembodiments, a member of a polypeptide class or family shows significantsequence homology or identity with, shares a common sequence motif(e.g., a characteristic sequence element) with, and/or shares a commonactivity (in some embodiments at a comparable level or within adesignated range) with a reference polypeptide of the class; in someembodiments with all polypeptides within the class). For example, insome embodiments, a member polypeptide shows an overall degree ofsequence homology or identity with a reference polypeptide that is atleast about 30-40%, and is often greater than about 50%, 60%, 70%, 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includesat least one region (e.g., a conserved region that may in someembodiments be or comprise a characteristic sequence element) that showsvery high sequence identity, often greater than 90% or even 95%, 96%,97%, 98%, or 99%. Such a conserved region usually encompasses at least3-4 and often up to 20 or more amino acids; in some embodiments, aconserved region encompasses at least one stretch of at least 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. Insome embodiments, a useful polypeptide may comprise or consist of afragment of a parent polypeptide. In some embodiments, a usefulpolypeptide as may comprise or consist of a plurality of fragments, eachof which is found in the same parent polypeptide in a different spatialarrangement relative to one another than is found in the polypeptide ofinterest (e.g., fragments that are directly linked in the parent may bespatially separated in the polypeptide of interest or vice versa, and/orfragments may be present in a different order in the polypeptide ofinterest than in the parent), so that the polypeptide of interest is aderivative of its parent polypeptide.

The term “reference” as used herein describes a standard or controlrelative to which a comparison is performed. For example, in someembodiments, an agent, animal, individual, population, sample, sequenceor value of interest is compared with a reference or control agent,animal, individual, population, sample, sequence or value. In someembodiments, a reference or control is tested and/or determinedsubstantially simultaneously with the testing or determination ofinterest. In some embodiments, a reference or control is a historicalreference or control, optionally embodied in a tangible medium.Typically, as would be understood by those skilled in the art, areference or control is determined or characterized under comparableconditions or circumstances to those under assessment. Those skilled inthe art will appreciate when sufficient similarities are present tojustify reliance on and/or comparison to a particular possible referenceor control.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention provides technologies for sterilization of peptidecompositions. In some embodiments, disclosed methods are particularlyapplicable to peptide solutions with high viscosity and/or stiffness. Insome embodiments, the present disclosure defines particular peptidesolutions that may be sterilized by autoclave treatment. In someembodiments, the present disclosure defines particular peptide solutionsthat may not be amenable to filtration unless and until treated so as toalter their rheological properties. In some embodiments, the presentdisclosure provides technologies that may temporarily reduce peptidesolution viscosity and/or stiffness sufficiently to permit filtration.In some embodiments, the present disclosure teaches technologies forfacilitating handling, processing, and/or filtration of certain peptidesolutions, for example by applying high shear stress that modifyrheological properties thereof.

Peptides and Peptide Compositions

In accordance with one or more embodiments, peptide compositions towhich teachings of the present disclosure may be compositions ofamphiphilic peptides having about 6 to about 200 amino acid residues. Incertain embodiments, a relevant peptide may have a length of at leastabout 7 amino acids. In certain embodiments, a peptide may have a lengthof between about 7 to about 17 amino acids. In certain embodiments, apeptide may have a length of at least 8 amino acids, at least about 12amino acids, or at least about 16 amino acids.

In some embodiments, as is understood in the art, an amphiphilicpolypeptide is one whose sequence includes both hydrophilic amino acidsand hydrophobic amino acids. In some embodiments, such hydrophilic aminoacids and hydrophobic amino acids may be alternately bonded, so that thepeptide has an amino acid sequence of alternating hydrophilic andhydrophobic amino acids. In some embodiments, such a peptide has anamino acid sequence that is or comprises repeats of Arg-Ala-Asp-Ala(RADA); in some embodiments, such a peptide has an amino acid sequencethat is or comprises repeats of Lys-Leu-Asp (KLD); in some embodiments,such a peptide has an amino acid sequence that is or comprises repeatsof Ile-Glu-Ile-Lys (IEIK).

In some embodiments, a peptide for use in accordance with the presentdisclosure, may generally be self-assembling, and/or may exhibit abeta-sheet structure in aqueous solution under certain conditions.

In some embodiments, a peptide for use in accordance with the presentdisclosure has an amino acid sequence as found in the commercial productknown as PuraMatrix®, i.e., has the amino acid sequenceArg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala (i.e.,RADA16, aka [RADA]4; SEQ ID NO:1). In some embodiments, a peptide foruse in accordance with the present disclosure has an amino acidsequence: Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu (i.e., KLDL12,aka [KLDL]3, aka KLD12; SEQ ID NO:2). a peptide for use in accordancewith the present disclosure has an amino acid sequence:Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile (i.e., IEIK13, aka(IEIK)3I; SEQ ID NO:3).

In some embodiments, peptide compositions to which the presentdisclosure may be relevant are those characterized by certainrheological properties. In some embodiments, relevant rheologicalproperties may be or include loss modulus, stiffness, rheologicalrecovery time, storage modulus, viscosity, yield stress, etc. In someembodiments, rheological properties are assessed via measurement; insome embodiments, one or more rheological properties may be assessed viavisual observation.

In certain embodiments, storage modulus and stiffness have a positivecorrelation; in general, those of ordinary skill appreciate that higherstorage modulus is related to higher stiffness.

In some embodiments, a high viscosity peptide composition ischaracterized by a storage modulus within the range of about 300 toabout 5,000 Pa at 1 rad/sec of frequency and 1 Pa of oscillation stress.

In some embodiments, a peptide composition for use in accordance withthe present invention has a peptide concentration within the range ofabout 0.01% to about 10%.

In some embodiments, a peptide composition to which one or more of themethodologies described herein is applied is of a commercial-scalevolume.

In some embodiments, a peptide composition to which one or more of themethodologies described herein is applied is one that has been storedfor a period of time. In some embodiments, a peptide composition hasbeen stored in a pressure vessel.

In some embodiments, a peptide composition to which one or moremethodologies described herein is applied is then stored, for example,in a reservoir vessel prior to packaging.

Improving Properties

The present disclosure appreciates that preparation and/or handling ofcertain peptide compositions (e.g., particularly compositions of certainself-assembling peptides and/or of high peptide concentrations) has beencomplicated by difficulties related, for example, to high viscosityand/or stiffness. The present disclosure particularly demonstrates thatcertain peptide compositions are not amenable to filtration, and inparticular to filtration through sterilizing filters.

The present disclosure further appreciates that filtration challengescan complicate or preclude sterilization of such peptide compositions.The present disclosure provides technologies that permit filtration ofcertain peptide compositions and/or otherwise permit sterilization.

Autoclave Treatment

Autoclave treatment is a conventional sterilization method that involvessubjecting materials to high pressure saturated steam at 121° C. It isgenerally understood in the art that application of high heat, such asis involved in autoclave treatment, can degrade peptides.

The present disclosure surprisingly demonstrates that certain peptidecompositions are stable to heat treatment, and particularly to autoclavetreatment. Among other things, the present disclosure demonstrates thatsuch peptide compositions may be sterilized with the autoclavetreatment. In some embodiments, such compositions may be sterilized byheat treatment at about 121° C. for about 25 minutes.

In some embodiments, peptide compositions that may be subjected to heattreatment, and/or to autoclave treatment are IEIK13 compositions. Insome such embodiments, IEIK13 compositions have a concentration withinthe range of about 0.01% to about 10%

In some embodiments, peptide compositions that may be subjected to heattreatment and/or to autoclave treatment are KLD12 compositions. In someembodiments, however, KLD12 compositions are not subjected to autoclavetreatment in accordance with the present invention.

In some embodiments, RADA16 compositions are not subjected to autoclavetreatment in accordance with the present invention.

Without wishing to be bound by any particular theory, the presentdisclosure proposes that the stability of certain IEIK13 compositions toheat treatment such as autoclave treatment may be attributable, at leastin part, to the absence of aspartic acid (Asp, D) in compositions, whileRADA16 and KLD12 have aspartic acids.

In some embodiments, peptide compositions that can appropriately besubjected to heat treatment such as autoclave treatment in accordancewith the present invention are characterized by resistance todegradation when exposed to such treatment and/or by stability ofrheological properties (e.g., viscosity and/or stiffness) when subjectedto such treatment. In accordance with the present disclosure, peptidecompositions of interest may be exposed to heat treatment such asautoclave treatment, and one or more properties of the composition(e.g., peptide degradation and/or one or more rheological properties)can be assessed, for example before and after treatment, so thatappropriateness of sterilizing such composition via autoclave treatmentmay be determined (see, e.g., Example 2).

Rheological Property Alteration

The present disclosure demonstrates that certain peptide compositionscan be rendered amenable to filtration via exposure to treatment thatalters one or more rheological properties (e.g., that alters viscosityand/or stiffness).

In some particular embodiments, rheological property alteration isachieved by exposure to shear stress.

Without wishing to be bound by any particular theory, the presentdisclosure proposes that subjecting peptide compositions as describedherein to high shear stress can disrupt self-assembled structures. Thepresent disclosure further proposes that recovery time may representthat required for such structures to re-form.

In some embodiments, shear stress applied to peptide solutions may be atleast about 20 Pa. In some embodiments, shear stress applied to peptidesolutions may be at least about 30 Pa. In some embodiments, shear stressapplied to peptide solutions may be at least about 40 Pa. In someembodiments, shear stress applied to peptide solutions may be at leastabout 50 Pa. In some embodiments, shear stress applied to peptidesolutions may be at least about 60 Pa. In some embodiments, shear stressapplied to peptide solutions may be at least about 60 Pa. In someembodiments, shear stress applied to peptide solutions may be at leastabout 80 Pa. In some embodiments, shear stress applied to peptidesolutions may be at least about 90 Pa. In some embodiments, shear stressapplied to peptide solutions may be at least about 100 Pa. In someembodiments, the amount of shear stress may be at least about 30˜100 Pa,for example, in view of the yield stress of RADA16 2.5%, IEIK13 1.5% and2.5% and KLD12 2.5% noted above.

In some embodiments, viscosity of peptide solutions may dropsignificantly with shear stress. In some embodiments, viscosity ofpeptides solutions may drop at least 10% with shear stress. In someembodiments, viscosity of peptides solutions may drop at least 30% withshear stress. In some embodiments, viscosity of peptides solutions maydrop at least 50% with shear stress. In some embodiments, viscosity ofpeptides solutions may drop at least 70% with shear stress. In someembodiments, viscosity of peptides solutions may drop at least 90% withshear stress.

In some embodiments, the rheological property alteration is temporary.In some embodiments, the peptide composition is characterized byrheological recovery characteristics. For example, in some embodiments,such compositions are characterized in that one or more of theirrheological properties are restored within a time period within a rangeof about 1 min to about 48 hours.

In some embodiments, rheological restoration is considered to beachieved when one or more rheological properties returns to a level atleast 20% of its initial value.

In some embodiments, rheological restoration is considered to beachieved when the change observed in one or more rheological propertiesupon application of shear stress is at least 30% reversed.

In some embodiments, peptide compositions may recover their storagemodulus after application of shear stress. In some embodiments, peptidesolutions may recover about 0.1 to 100% of their original storagemodulus in 1 min. In some embodiments, peptide solutions may recoverabout 0.1 to 10% of their original storage modulus in 1 min. In someembodiments, peptide solutions may recover about 20 to 100% of theiroriginal storage modulus in 20 min. In some embodiments, peptidesolutions may recover about 20 to 60% of their original storage modulusin 20 min.

In some embodiments, peptide solutions may recover their viscosity overtime after filtration. In some embodiments, peptide solutions mayrecover about 0.1 to 30% of their original viscosity in 1 min. In someembodiments, peptide solutions may recover about 0.1 to 100% of theiroriginal viscosity in 1 min. In some embodiments, peptide solutions mayrecover about 20 to 100% of their original viscosity in 20 min. In someembodiments, peptide solutions may recover about 20 to 60% of theiroriginal viscosity in 20 min.

The present disclosure specifically exemplifies appropriate adjustmentof rheological properties of certain peptide compositions uponapplication of shear stress (e.g., specifically upon passage through aneedle, for example of particular structure) (see Example 4). Theresults presented in this Example show a logarithmic increase of storagemodulus from 1 minute after injection, as shown in FIG. 10 for RADA16,FIG. 11 for KLD12, and FIG. 12 for IEIK13.

Among other things, the present disclosure provides methodologies inaccordance with which one or more certain peptide compositions aresubjected to high shear stress so that one or more of their rheologicalproperties is adjusted (e.g., viscosity is decreased) to an appropriatelevel so that the composition(s) become amenable to filtration, and insome embodiments to sterilizing filtration, and the composition(s) aresubjected to such filtration, within a time period after the subjectingto shear stress selected so that filtration occurs while the rheologicalproperties remain adjusted (e.g., before significant or completerestoration of such propert(ies) has occurred).

In general, as described herein, shear stress may be applied byapplication of a peptide composition to (and/or passage of a peptidecomposition through) a shear-thinning unit. In some embodiments, ashear-thinning unit is or comprises a needle, a membrane, and/or ascreen. In some embodiments, a plurality of individual shear-thinningunits is utilized, for example so that high-throughput filtration can beachieved.

In some embodiments, the present invention provides devices andmethodologies that can achieve filtration of peptide compositions on acommercial scale.

Needle as a Shear-Thinning Unit

In some non-limiting embodiments, shear stress may be applied byinjection through one or more needles. Thus, in some embodiments, one ormore needles may be used as a shear-thinning unit.

In some embodiments, a needle may be at least about 1 mm long. In someembodiments, a needle may be at least about 2 mm long. In someembodiments, a needle may be at least about 5 mm long. In someembodiments, a needle may be at least about a 10 mm long. In someembodiments, a needle may be at least about 15 mm long. In someembodiments, a needle may be at least about 20 mm long. In someembodiments, a needle may be at least about 30 mm long. In someembodiments, a needle may be at least about 40 mm long. In someembodiments, a needle may be at least about 50 mm long.

In some embodiments, a needle may have a gauge within a range of about20 to about 34. In some embodiments, a needle may have a gauge within arange of about 25 to about 34. In some embodiments, a needle may have agauge of about 27 to about 34.

FIG. 5 discloses one non-limiting embodiment of a sterilization devicein accordance with one or more non-limiting embodiments. As depicted,peptide composition (e.g., viscous solution of a self-assemblingpeptide) (left) may be transferred to the first syringe with a needle,injected to the second syringe (right), and then filtered.

Membrane as a Shear-Thinning Unit

In some embodiments, a shear-thinning unit utilized to apply shearstress to a peptide composition as described herein may be a device orentity characterized by micro- or nano-pores. FIG. 13 depicts onenon-limiting embodiment of a sterilization device in accordance with oneor more non-limiting embodiments of the present invention. As depicted,a peptide solution (e.g., a viscous solution of a self-assemblingpeptide) may be transferred to a dispensing syringe (or a pressurevessel), delivered to a first chamber with pores for shear stress, andthen filtered in the second chamber. As will be understood by thoseskilled in the art, diameter size of membrane may vary depending on theamount of peptide solution.

In some embodiments, pore size of a shear-thinning unit may be about0.45 μm to 120 μm. In some embodiments, pore size of a shear-thinningunit may be about 1 μm to 100 μm. In some embodiments, pore size of ashear-thinning unit may be about 3 μm to 80 μm. In some embodiments,pore size of a shear-thinning unit may be about 4 μm to 50 μm.

Screen as a Shear-Thinning Unit

In some embodiments, a shear-thinning unit may have micro- ornano-holes. In some embodiments, holes may be patterned or drilled on aplate whose thickness may be about 10 μm to 10 mm in some embodiments.FIG. 15 depicts one non-limiting embodiment of a sterilization device inaccordance with one or more non-limiting embodiments of the presentdisclosure. A shear-thinning unit may be inserted into the firstfiltering chamber shown FIG. 13.

In some embodiments, holes in an embodiment of a shear-thinning unitdescribed herein may have a largest dimension within the range of aboutmay be about 0.5 μm to 200 μm. In some embodiments, such dimension maybe within the range of about 0.5 μm to 100 μm. In some embodiments, suchdimension may be within the range of about 0.5 μm to 80 μm. In someembodiments, In some embodiments, such dimension may be within the rangeof about 0.5 μm to 50 μm.

In some embodiments, a shear-thinning unit of this embodiment may have apitch between holes within the range of about 5 μm to about 10 mm.

In some embodiments, shear-thinning unit may be made, in whole or inpart, of a material selected from the group consisting ofstainless-steel, tungsten, titanium, similar metal, silicon, ceramic orplastic materials, and combinations thereof.

Applications

In some embodiments, peptide compositions to which technologiesdescribed herein are applied are then utilized in one or moreapplications that involve biological cells, tissues, or organisms (e.g.,so that sterilized compositions are of particular utility).

As is known in the art, certain peptide compositions (e.g., certaincompositions of self-assembling peptides) have proven to be particularlyuseful as matrices for cell growth in vivo and/or in vitro, and/or asvoid fillers, hemostats, barriers to liquid movement, wound healingagents, etc. In some embodiments, such compositions form peptidehydrogels with one or more desirable characteristics (e.g., pore and/orchannel size, strength, deformability, reversibility of gel formation,transparency, etc).

Those skilled in the art, reading the present disclosure, willimmediately appreciate its usefulness in a variety of contexts in whichsuch peptide compositions, including gel compositions and especiallyincluding reversibly gelling compositions, are employed. Of particularinterest are in vivo applications (e.g., surgical applications or otherapplications, particularly that permit or benefit from delivery via acannula-type device, such as a needle, through which composition may beadministered or applied).

EXEMPLIFICATION Example 1: Filtration of High Viscous Peptide Solutions

The present Example describes, among other things, rheologicalproperties of various peptide compositions (i.e., specifically ofcompositions of self-assembling peptides), and demonstrates significantvariability of parameters such as viscosity, storage modulus (e.g.,stiffness), loss modulus, and yield stress for different peptides and/orfor different concentrations of the same peptide. The Example alsodemonstrates that certain of these solutions are not readily amenable tofiltration. In particular, the Example demonstrates that high viscositysolutions of such peptides present challenges for filtrationtechnologies. Rheological properties were determined for a variety ofpeptide solutions. Specifically, solutions of RADA16, IEIK13, and KLD12per prepared at concentrations indicated below in Table 1. As can beseen, in general, higher concentration solutions showed higher maxviscosity. Furthermore, peptides of different sequence showed differentmax viscosities in solutions of the same concentration. For example 2%KLD12, 2.5% KLD12, and 1.5% IEIK13 solutions have 2, 3.4, and 3.2 timeshigher maximum viscosities than 2.5% RADA16, respectively.

TABLE 1 Rheological properties of peptide solutions at selectedconcentrations Storage Max. Modulus Loss Modulus Viscosity Conc. (G′)*(G″)* Yield Stress (max η′) Peptides (%) (Pa) (Pa) (Pa)*^(,#) (Pa ·s)*^(,#) RADA16 1 74 12 15.9 2.3 1.5 158 30 20.0 3.4 2 217 39 31.6 4.02.5 352 53 50.1 5.6 IEIK13 1 719 77 39.8 12.9 1.5 1092 94 50.1 18.0 21708 138 — — 2.5 2213 174 100 40.2 KLD12 1 140 25 25.1 2.0 1.5 292 4639.8 7.1 2 573 63 79.4 11.0 2.5 846 93 100 19.0 *at 1 Pa of oscillationstress ^(#)Maximum viscosity data was adapted in viscosity plots at therange of measured stress.

Each of the peptide solutions listed in Table 1 was subjected tofiltration through a 0.2 μm Nalgene syringe filter with 25 mm celluloseacetate membranes. The 1% and 1.5% KLD12 solutions (which, as can beseen, are characterized by relatively low concentration, viscosityand/or stiffness) passed successfully through the filter. By contrast,the 2% and 2.5% KLD12 solutions and 1.5% IEIK13 solutions (which, as canbe seen, are characterized by relatively high concentration, viscosityand/or stiffness) could not be passed successfully through the filter;instead, the filter burst.

Example 2: Autoclave Treatment of Peptide Solutions

The present Example demonstrates that some peptide compositions (i.e.,specifically compositions of self-assembling peptides as describedherein) are surprisingly stable to heat treatment. In particular, thisExample demonstrates that certain peptide compositions maintain a stablemolar mass even upon application of autoclave treatment at 121° C. for25 minutes. The present Example therefore establishes that suchcompositions can successfully be sterilized through application of highheat (e.g., autoclave) technologies. The Example simultaneouslydemonstrates, however, that certain peptide compositions are not stableto such treatment.

FIGS. 1-3 present results of autoclave treatment for certaincompositions of RADA16, IEKI13, and KLD12, respectively.

The measured molar mass of RADA16, prior to autoclave treatment, was1712, which matches its calculated molar mass. However, the mass specanalysis demonstrated that RADA16 was degraded during the autoclavetreatment, thereby demonstrating that this technique cannot be used forsterilization of such a RADA16 composition.

The measured molar mass of IEIK13, prior to autoclave treatment, was1622, which also matches its calculated molar mass. Mass spec analysisdemonstrated that IEIK13 was not degraded after the autoclave treatment,thereby demonstrating that this technique can usefully be employed forsterilization of such an IEIK13 composition.

The measured molar mass of KLD12, prior to autoclave treatment, is 1467,which matches its calculated molar mass. KLD12 was partially degradedduring autoclave treatment. As KLD12 was degraded during autoclavetreatment, it was determined that autoclave treatment is not a preferredtechnique for sterilization of such KLD12 compositions; a conventionalfiltration approach to sterilization was carried out on KLD12 at severalconcentrations of peptide.

Rheological properties of certain peptide compositions were determinedbefore and after autoclaving. The data are shown in FIG. 4. As can beseen, autoclaved IEIK13 surprisingly exhibited almost identicalrheological strength as non-autoclaved IEIK13, while RADA16 displayed adramatic decrease of rheological strength.

Autoclave treatment may be used for sterilization of IEIK13 compositionsas described herein, but should be avoided for RADA16 compositions.

Example 3: Rheological Properties of Peptide Compositions withApplication of Shear Stress

The present Example demonstrates that applied shear stress may decreaseviscosity and/or stiffness of certain peptide solutions, and furthermoredemonstrates that such decrease in viscosity and/or stiffness can renderthe compositions amenable to various and/or processing technologies(e.g., filtration) to which the compositions are not amenable absentsuch treatment.

Shear Flow Test

Shear flow tests were performed on peptide solutions using a rheometer(DHR-1, TA Instruments) with 20 mm plates. Results are shown in FIG. 9for 2.5% RADA16 solutions and FIG. 10 for 1.5% IEIK13 solutions. As canbe seen, both 2.5% RADA16 and IEIK13 1.5% solutions showed a typicalshear thinning properties. That is, as shear rate increased, theirviscosities were dramatically dropped. As shear rate increased, shearstress immediately increased, and then slightly decreased when viscosityreached a plateau. The yield stress was about 40 Pa for 2.5% RADA16solution and about 60 Pa for 1.5% IEIK13 solution.

Viscosity Recovery

The viscosity recovery times of RADA16 and IEIK13 solutions wereevaluated after application of high shear stress. Using a DHR-1rheomether (TA Instruments), viscosity changes of 2.5% RADA16 and 1.5%IEIK13 solutions were measured with flow tests at 0.005 l/sec of shearrate after applying 1000 l/sec of shear rate to samples for 1 min.RADA16 and IEIK13 solutions showed a typical thixotropic behavior, whichmeans their viscosity were slowly recovered. Without wishing to be boundby any particular theory, we propose that rheological property recoverytimes for these solutions may be based on re-assembly of peptidemolecules into structures (e.g., nano-fibers) in the solutions. Completereassembling times of 2.5% RADA16 and 1.5% IEIK13 solution were about 12to 48 hours. The results are shown in FIG. 11 for 2.5% RADA16 solutionand FIG. 12 for 1.5% IEIK13 solution.

Storage Modulus Recovery

The percentages of recovery back to the original storage modulus at 1min and 20 min after injection peptide compositios through a 30 gaugeneedle are listed in Table 2. The recovery rate of IEIK13 (specifically,of a 2.5% IEIK13 solution) was the fastest among the peptide solutions,showing 100% recovery to the original storage modulus in 20 min. KLD12was the slowest among those tested to recover; it showed only 23%recovery to the original storage modulus in 20 min (for 2.5%). In somenon-limiting embodiments, it may take about 12 to 48 hours for fullrecovery to an original modulus after passage through a needle (e.g.,injection).

TABLE 2 Recovery to the original storage modulus at 1 min and 20 minafter injection through 30-gauge needle 1% 2.5% 1 min 20 min 1 min 20min Before after after Before after after injection injection injectioninjection injection injection RADA16 Storage modulus  74 Pa  6.8 Pa 40Pa 352 Pa 67 Pa 196 Pa (Pa) Recovery % to — 9.2% 54% — 19% 56% theoriginal modulus KLD12 Storage modulus 140 Pa 0.68 Pa 59 Pa 846 Pa 57 Pa196 Pa (Pa) Recovery % to — 0.5% 42% — 6.7%  23% the original modulusIEIK13 Storage modulus — — — 2213 Pa  632 Pa  2248 Pa  (Pa) Recovery %to — — — — 29% 100%  the original modulus

Rheological measurements were performed for RADA16 and IEIK13 solutionsafter injecting them through 30 gauge needles. The results showed alogarithmic increase of storage modulus from 1 minute after injection.The results are shown in FIG. 6 for RADA16, FIG. 7 for KLD12, and FIG. 8for IEIK13.

Example 4: A Needle as a Shear-Thinning Unit

The present Example describes a filtration process for peptidecompositions (specifically, of self-assembling peptides as describedherein) using a needle as a shear-thinning unit. In particular, thepresent Example demonstrates that application of appropriate shearstress (e.g., via passage through a shear-thinning unit) can alterrheological properties of the composition (e.g., can reduce viscosityand/or stiffness, etc) so that it can successfully be passed through afilter such as, for example, a sterilizing filter).

FIG. 5 depicts one non-limiting embodiment of a sterilization device inaccordance with the present disclosure. As depicted, the device includesa first syringe that applies sheer stress to the composition sufficientto alter its rheological properties such that it successfully passesthrough a second syringe that is fitted with a membrane filter ofappropriate pore size to achieve sterilization of the composition.Specifically, the depicted device includes a first syringe with a 30gauge needle (0.3 mm×25 mm, Endo irrigation needle with double sidevent, Transcodent, Germany) (middle) and a second syringe with amembrane filter (right). A viscouse 2.5% KLD12 solution (left) wastransferred to the first syringe, and was then injected into the secondsyringe (right) and then filtered through the membrane filter. Usingthis method, 2.5% KLD12 solutions were successfully filtered.

Example 5: High Throughput Shear-Thinning Unit

The present Example describes certain shear thinning units. Theprinciple of operation is like that for the first needle describedabove. Specifically, each shear-thinning unit applies shear stressappropriate and sufficient to adjust one or more rheological propertiesof an applied peptide composition so that the composition becomesamenable to filtration, and specifically to filtration through asterilizing filter. In some embodiments, multiple needles or equivalentsmay be used as a shear-thinning unit.

Membrane Filter

This Examples demonstrates use of a with membrane filter (poresize >0.45 μm) as a shear-thinning unit. Viscous 2.5% KLD12 or 1.5%IEIK13 solutions may be transferred to a dispensing syringe (or apressure vessel), delivered to a first chamber with a shear-thinningunit (for example, pore size ranging from 0.45 μm to 120 μm), and thenfiltered through a filtering membrane (for example, pore size: 0.2 μm)in the second chamber.

To examine the effect of pore size in the shear-thinning unit onviscosity change of viscous peptide solutions, 2.5% KLD12 and 1.5% IEIKsolutions were passed through selected pore sizes, and their apparentviscosity changes were evaluated. 2.5% KLD solutions passed through theshear-thinning unit with the pore sizes of 41 μm, 20 μm, and 5 μm.Viscosity of the solutions was decreased enough to flow down when avessel containing it was flipped over. Though 2.5% KLD solutions thathad passed through the shear-thinning unit with the pore size of 120 μmwere slightly less viscous than pre-passage 2.5% KLD compositions, theyremained too viscous to flow down in the container-inversion test. Theviscosity of 1.5% IEIK13 solutions was reduced significantly when passedthrough a membrane with a pore size of 5 μm. Results are shown in FIG.14.

1.0% RADA16 solutions were studied for viscosity reduction with ashear-thinning unit (shown in FIG. 13). 1.0% RADA16 solution, whichshows shear thinning and thixotropic behavior, was passed through ashear-thinning unit at 50 psi of injection pressure. The solution showed1.4˜1.7 mL/min of output. The solution could not be passed through afilter (0.2 μm pore size) at 50 psi of injection pressure (i.e., withoutprior exposure to a shear-thinning unit). However, water, which is arepresentative Newtonian fluid, showed that output flow rate wasrelatively consistent. The results are shown in Table 3.

TABLE 3 Filtering abilities of the system shown in FIG. 13 for RADA16 1%solution and water. Output through Output through Output throughshear-thinning unit shear-thinning unit shear-thinning Output through(41 μm pore size) + (20 μm pore size) + unit (5 μm pore filter: 0.2 μmpore filter (0.2 μm filter (0.2 μm size) + filter (0.2 μm Materialssize* pore size)* pore size)* pore size)* Water (at 25 psi) 42 mL/min 35 mL/min  41 mL/min  33 mL/min RADA16  0 mL/min 1.4 mL/min 1.7 mL/min1.6 mL/min 1.0% (at 50 psi) *Diameters of shear-thinning units andfilter are 25 mm.

As demonstrated above, 2.5% KLD12 and 1.5% IEK13 solutions were not ableto be filtered through a 0.2 μm Nalgene syringe filter with 25 mmcellulose acetate membranes. 2.5% RADA16 is not usually amenable tofiltration through 0.2 μm membrane. 2.5% RADA16, 1.5% IEIK13, and 2.5%KLD12 solutions were able to be filtered after being exposed to ashear-thinning unit at 100 psi of injection pressure showing 3.8, 12.5,and 11.4 mL/min of output, respectively. The solutions were not able tobe filtered without the shear-thinning unit. A shear-thinning unit shownin FIG. 16 may be successfully utilized for sterilization and filtrationof viscous peptide solutions which are not easily filtered. The resultsare shown in Table 4.

TABLE 4 Filtering abilities filtering system shown in FIG. 13 for 2.5%RADA16, 1.5% IEIK13, 2.5% KLD12 solutions. Output through Output throughshear-thinning unit filter (0.2μ pore (5 μm pore size) + filter size) at100 psi.* (0.2 μm pore size) at 100 psi.* RADA16 2.5% 0 mL/min  3.8mL/min IEIK13 1.5% 0 mL/min 12.5 mL/min KLD12 2.5% 0 mL/min 11.4 mL/min*Diameters of membranes are 25 mm.

Screen

This Examples are demonstrates successful use of a screen with micro-and/or nano-holes as a shear-thinning unit. Viscous 2.5% KLD12 or 1.5%IEIK13 solutions may be transferred to a dispensing syringe (orchamber), injected to a first chamber that includes a shear-thinningunit with micro- and/or nano-holes, and then filtered through themembrane filter (pore size: 0.2 μm) in the second chamber. Instead ofsyringe for injection, a high pressure chamber may be used to deliver apeptide composition. Membrane size (e.g., diameter) and/or othercharacteristics (e.g., pore size, etc) may be selected to accommodateamount of peptide composition to be passed through it.

TABLE 5 Filtering abilities of the micro-hole screen system shown inFIG. 13 and FIG. 15 for 2.5% RADA16, 1.5% IEIK13, 2.5% KLD12 solutions.Output through Output through shear-thinning unit filter (0.2μ pore(screen with micro holes^(#)) + filter size) at 100 psi.* (0.2 μm poresize) at 100 psi.* RADA16 2.5% 0 mL/min 4.2 mL/min IEIK13 1.5% 0 mL/min5.0 mL/min KLD12 2.5% 0 mL/min 11.5 ml/min   *Diameters of membranes are25 mm. ^(#)hole size is 50 μm in diameter, pitch of holes is 450 μm, anddepth of holes is 500 μm.

What is claimed is:
 1. A method for sterilizing a liquid peptidecomposition whose sequence comprises a series of repeating units of IEIKcomprising subjecting the composition to autoclave treatment.
 2. Themethod of claim 1, wherein the method does not involve sterilizingfiltration.
 3. A method for sterilizing a liquid peptide compositionwhose sequence comprises a series of repeating units of IEIK comprisingsubjecting the composition to heat treatment
 4. The method of claim 3,the heat treatment performs at about 121° C. for about 25 min.
 5. Amethod for sterilizing a liquid peptide composition having an initialstorage modulus within the range of about 300 to about 5,000 Pa at 1rad/sec of frequency and 1 Pa of oscillation stress, the methodcomprising steps of: subjecting the liquid peptide composition to highshear stress so that storage modulus of the composition is temporarilyreduced to a level within a range of about 0.01% to 80% of the initialstorage modulus; and subjecting the composition to filtration while itsviscosity is at the reduced level.
 6. The method of claim 5, wherein thestep of subjecting the composition to high shear stress utilizes atleast one shear-thinning unit.
 7. The method of claim 6, wherein the atleast one shear-thinning unit is or comprises at least one needle. 8.The method of claim 7, wherein the at least one needle is at least 1 mmlong.
 9. The method of claim 7, wherein the at least one needle has agauge within the range of about 25 to about
 35. 10. The method of claim6, wherein the at least one shear-thinning unit is or comprises at leastone screen with micro- or nano-sized holes.
 11. The method of claim 10,wherein the micro- or nano-sized holes have a largest dimension within arange of about 0.5 μm to about 200 μm.
 12. The method of claim 10,wherein a pinch between holes is about 5 μm to about 10 mm.
 13. Themethod of claim 10, wherein the screen is made at least in part of amaterial selected from the group consisting of stainless-steel,tungsten, titanium, silicon, ceramic, plastic, and combination thereof.14. The method of claim 10, wherein thickness of the screen is about 10μm to about 10 mm.
 15. The method of claim 6, wherein the at least oneshear-thinning unit is or comprises at least one membrane with micro- ornano-sized pores.
 16. The method of claim 15, wherein the pores gave asize with a range of about 0.45 μm to about 120 μm.
 17. The method ofclaim 5, wherein the high shear stress is with a range of about 30 toabout 200 Pa.
 18. The method of claim 5, wherein the liquid peptidecomposition comprises RADA16, IEIK13, or KLD12.
 19. The method of claim5, wherein the liquid peptide composition is pressurized prior tofiltration.
 20. The method of claim 5, further comprising storing theliquid peptide composition under vacuum.